Protein markers for the diagnosis and prognosis of ovarian and breast cancer

ABSTRACT

Plasma samples of ovarian and breast cancer patients were used to search for markers of cancer, using two-dimensional gel electrophoresis and MALDI TOF mass spectrometry. Truncated forms of cytosolic serine hydroxymethyl transferase (cSHMT), T-box transcription factor 3 (Tbx3) and utrophin were aberrantly expressed in samples from cancer patients, as compared to samples from noncancer cases. Aberrant expression of proteins was validated by immunoblotting of plasma samples with specific antibodies to cSHMT, Tbx3 and utrophin. A cohort of 79 breast and 39 ovarian cancer patients, and 31 individuals who were either healthy or had noncancerous conditions was studied. We observed increased expression of truncated cSHMT, Tbx3 and utrophin in plasma samples obtained from patients at early stages of disease. The results indicate that cSHMT, Tbx3, utrophin and truncated forms thereof can be used as components of multiparameter monitoring of ovarian and breast cancer.

This application claims the benefit of U.S. Provisional Application No.60/834,309, filed Jul. 27, 2006.

FIELD OF THE INVENTION

The present invention is directed to the field of cancer detection anddiagnosis. More particularly, it is directed to new protein markers andtheir use in diagnosing and monitoring ovarian and breast cancer.

BACKGROUND OF THE INVENTION

Blood plasma is an easily accessible source of proteins which havediagnostic value, as it is in contact with practically all tissues inthe human body. Plasma proteome contains not only proteins whichfunction in or via plasma, e.g. albumin, immunoglobulins and cytokines,but also proteins which leak from tissues.¹ During tumor growth, damageof normal or tumor tissues leads to release of cellular proteins intoplasma. Currently, 196 proteins have been identified in human plasma,but up to 1175 distinct gene products may be present in human plasma.²

Plasma has been extensively explored in searches for markers oftumorigenesis. However, attempts to find a single reliable early markerof human breast or ovarian cancer have not been fully successful.Variability of molecular mechanisms governing tumor growth and itsspreading in the body indicates that an expression pattern of a fewmarkers may be more informative than a single marker.^(3,9,16) Thisprompts a search for proteins which change their expression in anaccessible diagnostic material, e.g. blood. Two-dimensional gelelectrophoresis (2D-GE), liquid chromatography and mass spectrometry, aswell as various array techniques have been used.³ Among describedmarkers, CA125, CA15-3, CEA, and RS/DJ1 proteins have been proposed asuseful markers to monitor breast cancer.^(3,4) CA125, apolipoprotein A1,transthyretin, inter-α trypsin inhibitor heavy chain H4, haptoglobin-1and kallikrein have been proposed as markers of ovarian cancer.⁵⁻⁸ Noneof the identified protein markers has been found to predict cancerappearance and development with a probability value close to 100%. Thiscan be explained by the presence of these proteins in normalnontransformed cells, as well as in tumor cells; in tumors, markerproteins change their activities or relocalize in cells.

A combination of a few markers was proposed to predict the appearanceand development of tumors with higher accuracy, as compared to use of asingle marker. The application of surface-enhanced laserdesorption/ionization mass spectrometry (SELDI) provides one means ofimplementing such a multiparameter approach.⁹ A combination ofidentified markers would be the most preferable solution for suchmultiparameter diagnostics. However, SELDI does not provide identitiesof proteins or peptides in identified mass peaks.

Cytoplasmic serine hydroxymethyl transferase (cSHMT) is one of the keyenzymes of one-carbon metabolism, the pathway which is altered incolorectal cancer.¹⁴ Increased expression of cSHMT has been found inmetastatic human breast carcinoma cells MDA-MB-435, as compared tononmetastatic cells.¹⁷ Whether cSHMT function is affected in breast andovarian cancers remains to be investigated. T-box transcription factor 3(Tbx3), on the contrary, is involved in development of mammary gland;mutations or lack of Tbx3 result in mammary hypoplasia, and even in lackof mammary glands.¹⁵ Tbx3 has been described as a potent inhibitor ofsenescence of neuronal cells and embryonal fibroblasts.¹⁸ Utrophin hasbeen described first as a protein involved in development ofneuromuscular junctions. However, utrophin detection in a variety ofcells has indicated its role in organization of connections betweencytoskeleton and transmembrane proteins. Utrophin has a number ofsplicing forms, and a variety of short isoforms have been described.¹³Utrophin has been detected in mammary ductal epithelium and in stroma.¹⁹Expressions of utrophin and its binding partner dystroglycan were foundto be reduced in breast adenocarcinomas.¹⁹ It is possible that thedecrease of utrophin in cells is accompanied by release of this proteininto the plasma. cSHMT, Tbx3 and utrophin are known to function insideof cells, and their appearance in plasma in truncated forms suggeststhat they were released from cells and subjected to a limitedproteolysis.

Herein is described the increased appearance of truncated forms ofcSHMT, Tbx3 and utrophin in plasma of patients with breast and ovariancancer. The use of these markers alone or, in a multiparameter approach,in combination with each other or in combination with other markersprovides a significant development and improvement in cancerdiagnostics.

SUMMARY OF THE INVENTION

The present invention is directed to new protein markers useful in thediagnosis and prognosis of ovarian and breast cancer. The termsexpression, aberrant expression, etc. in the present application areused to describe the presence, amount, level or relative level of thecSHMT, Tbx3 and utrophin proteins, or truncated forms thereof, in plasmaof cancer patients or patients without cancer (controls), except wherespecifically stated otherwise. More particularly, the invention isdirected to cSHMT, Tbx3 and utrophin and truncated forms thereof, theaberrant expression of which in plasma has now been shown to correlatewith increased incidence of ovarian and breast cancer. In a relatedvein, low levels of expression have now been associated with cancer-freepatients. In a related discovery, lower levels of these proteins incancer patients have now been correlated with increased time and chanceof survival and, in a related vein, aberrant expression has now beenlinked to low rates and decreased times of survival. The invention isdirected both to the new protein markers and to methods for detectingthe onset of ovarian and beast cancer and monitoring the progressthereof. The invention is further directed to antibodies raised againstthe new markers and which serve as a vital component of the intendeddiagnostic methods.

One aspect of the invention provides a method employing the markers fordiagnosis of ovarian and/or breast cancer. A plasma sample from apatient is tested for the presence and amounts of the markers, and theresults indicate the presence or absence of the cancer(s).

In another aspect of the invention, the markers of the invention areused in a prognostic application. A plasma sample from a patient withovarian and/or breast cancer is tested for the presence and amounts ofthe markers, and the results are correlated with duration and chance ofsurvival.

Yet another aspect of the invention is a kit comprising antibodiesraised against one or more of the markers, which kit can be used as adiagnostic and prognostic tool in connection with ovarian and/or breastcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic representation of the method for identifying theprotein markers of the present invention.

FIG. 1B shows a typical 2D gel illustrating the quality of proteinresolution.

FIG. 1C shows gels showing the areas of migration of the marker proteinsin noncancerous, ovarian cancer and breast cancer patients.

FIGS. 2A-C show various aspects of the detection of cSHMT in plasmasamples by immunoblotting.

FIGS. 3A-C show various aspects of the detection of Tbx3 in plasmasamples by immunoblotting.

FIGS. 4A-C show various aspects of the detection of utrophin in plasmasamples by immunoblotting.

FIGS. 5A-F are graphic depictions of the expression of cSHMT, Tbx3 andutrophin in plasma of patients with various stages of cancer.

FIG. 6 is a graphic depiction of the correlation between expression ofcSHMT in plasma and survival time of ovarian cancer patients.

FIG. 7 is a graphic depiction of the correlation between expression ofcSHMT in plasma and overall survival of ovarian cancer patients.

FIG. 8 is a graphic depiction of the correlation between expression ofTbx3 in plasma and overall survival of breast cancer patients.

FIG. 9 is a graphic depiction of the correlation between expression ofutrophin in plasma and overall survival of breast cancer patients.

DETAILED DESCRIPTION OF THE INVENTION Material and Methods Patient Group

Samples were collected from 79 breast cancer patients, 39 ovarian cancerpatients, 28 patients with nonmalignant processes of pelvis and breast,and 3 healthy individuals (Table 1). Clinical, instrumental,histopathological diagnostics and treatment of patients were performedat the Lviv Regional Oncology Center. The information about patients wasdeposited in the Cancer Register database of the Lviv Regional OncologyCenter. Sample collection was performed with consent of the patients, inaccordance with the rules for handling of medical information andmedical samples, and under supervision of the Ethical Committee of theLviv National Medical University. Staging of patients was performedaccording to the Federation of Gynaecologic Oncologists (FIGO) and TNM(9^(th) edition) classifications. Samples were coded immediately aftercollection, and coding was preserved throughout the study untilconclusive proteomics and immunoblotting data had been generated.

Sample Preparation

Plasma protein samples were prepared from cubital venous blood; bloodwas collected into tubes with 0.3% sodium citrate solution. Blood wascollected in the morning, before breakfast and any physical activities.Shortly after collection (10-15 min) blood was centrifuged for 10 min at3000 rpm. Aliquots of plasma were transferred into 1.5 ml tubes andproteins were precipitated on ice with 96% ethanol (ratio 1:3;plasma:ethanol). Other ratios of plasma:ethanol (1:1, 1:5, 1:10), aswell as precipitation with acetone, resulted in significantly lessefficient precipitation and/or recovery of plasma proteins. The pelletwas obtained by centrifugation (20 min, 13000 rpm, +4° C.). Supernatantwas discarded, and plasma-precipitated proteins were air-dried andstored at room temperature in closed tubes until further analysis. Weobserved that precipitated plasma proteins prepared according to thisprotocol can be stored for up to two years without deterioration ofprotein recovery and resolution in two-dimensional gel electrophoresis.

Two-Dimensional Gel Electrophoresis

Samples were dissolved in 2D-GE buffer (8 M urea, 4% CHAPS, 0.5% DTT,IPG buffer pH 3-10). Samples (100 μg of protein) were subjected toisoelectric focusing (IEF) using 18 cm linear IPGDry strips with a pHrange of 3-10 (Amersham Biosciences). Samples were loaded by the in-gelrehydration technique, with active loading during the last 3 h. IEF wasperformed in an IPGphor (Amersham Biosciences, Uppsala, Sweden) usingthe following protocol: rehydration, 10 h, then 50 V, 3 h; 1,000 V, 1 h;8,000 V, 10 h; or until the total volt-hours reached 50,000. After IEF,strips were equilibrated in 50 mM Tris-HCl pH 8.8, 6 M urea, 2.0% SDS,30% glycerol with 1% DTT for 10 min, and then for 10 min in the samebuffer containing 4% iodoacetamide instead of DTT. Equilibrated stripswere placed on top of 12% polyacrylamide gels and fixed with 0.5%agarose in 62.5 mM Tris-HCl pH 6.8, 0.1% SDS. Sodium dodecylsulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed in aDalt-Six, following the manufacturer's recommendations (constant power50 W, for 6 to 8 h; Amersham Biosciences). Gels were fixed in 10% aceticacid and 20% methanol for 10-12 h. Proteins were detected by silverstaining as previously described.^(10,11)

Image Analysis

Silver-stained gels were scanned in an ImageScanner with the MagicScan32software and analyzed with calculation of volumes of spots by theImageMaster 2D Elite software (Amersham Biosciences). Protein spotsdifferentially expressed in cancer and noncancer patients wereconsidered for protein identification. The statistical significance ofchanges was evaluated using the ImageMaster 2D Elite software.¹⁰

Protein Identification

Protein spots were excised from gels, destained and subjected to in-geldigestion with trypsin (modified, sequence grade porcine; Promega,Madison, Wis., USA), as described.^(10,11) Tryptic peptides wereconcentrated and desalted on a nanocolumn. Peptides were eluted with 50%acetonitrile containing α-cyano-4-hydroxycinnamic acid as the matrix,applied directly onto the metal target and analyzed by matrix-assistedlaser desorption/ionization time of flight mass spectrometry (MALDI TOFMS) on a Bruker Autoflex MALDI TOF mass spectrometer (Bruker Daltonics,Bremen, Germany). Peptide spectra were internally calibrated usingtrypsin autolytic peptides. To identify proteins, we searched the NCBIdatabase using the ProFound search engine(http://65.219.84.5/service/prowl/profound.html). One miscut and partialoxidation of methionine were allowed. Probability of identification wasevaluated according to the probability value (Z value) and sequencecoverage. Comparison of the experimental pI and Mr values of proteins totheir theoretical values was also considered.

Immunoblotting

For immunoblotting of cSHMT, Tbx3 and utrophin, aliquots of plasma,which were prepared as for 2D-GE, were dissolved in an SDS-PAGE samplebuffer and 0.5-1.0 μg of plasma protein was subjected to 1D SDS-PAGE.After transfer to nitrocellulose membrane, proteins of interest weredetected with specific antibodies: cSHMT with D-20 antibody (sc-25060,which recognizes an epitope within the C-terminal part of cSHMT), Tbx3with C-20 antibody (sc-17872, which recognizes an epitope within theinternal region of Tbx3) and utrophin with N-19 antibody (sc-7460, whichrecognizes an epitope within the N-terminal part of utrophin). Allantibodies were from Santa Cruz Biotechnology, Santa Cruz, Calif., USA.Protein loading was controlled by internal expression of plasmaimmunoglobulins, and by reprobing the same membranes with anti-human IgG(ab6858, Abcam Ltd, Cambridge, UK). Proteins of interest were detectedby ECL as previously described.^(10,11) Intensity of detected bands wasmeasured using a CCD camera (LAS-1000 CH, Fuji, Japan), and dedicatedprograms (Scion Image beta 4.0.2, Scion Corp., Frederick, Md., USA, andAIDA, Raytest IMG, Sprockhofel, Germany). Intensities of IgG bands weremeasured after reprobing with antihuman IgG the same membranes whichwere first probed with anti-cSHMT, anti-Tbx3 and antiutrophinantibodies.

Relative expressions of cSHMT, Tbx3 and utrophin in plasma werecalculated as the ratio of values of a signal from cSHMT-, Tbx3- orutrophin-specific bands to IgG signal. For definition of weak, mediumand strong expression, intensities of specific bands in immunoblots withantibodies to cSHMT, Tbx3 and utrophin were considered. Then, averageratio of intensities of specific protein to IgG bands in the same samplewere calculated to define weak, medium and strong specific proteinbands. As IgG bands were on average stronger, as compared to intensitiesof specific proteins, consistently shorter (though equal for all blots)exposure time for IgG immunoblotting was used. This explains the valuesof the ratios. These ratios were taken as cutpoints for representationof relative expression of cSHMT, Tbx3 and utrophin. The cutpoints weredefined as follows. For cSHMT: weak expression, less than 25%; mediumexpression, 26%-50%; strong expression, more than 50%. For Tbx3: weakexpression, less than 40%; medium expression, 41%-60%; strongexpression, more than 61%. For utrophin: weak expression, less than 40%;medium expression, 41%-70%; strong expression, more than 71%. Theseratios were used for the correlation analysis of expression of studiedproteins in plasma with stages of disease.

Clinical information was available from the Department of Oncology andRadiology of the Lviv National Medical University at the Lviv OncologyCenter. Results of the correlation analysis were expressed as apercentage of cases with weak, medium or strong expression of studiedproteins. Sensitivity was calculated as a ratio of number of cases withdetection of specific aberrantly expressed proteins to total number ofcancer cases. Specificity was calculated as a ratio of cases withnegative detection of specific aberrantly expressed proteins to totalnumber of control noncancer cases.

For detection of cSHMT, Tbx3 and utrophin in cultured cells, weperformed immunoblotting assays with whole-cell extracts as describedabove for plasma samples. To evaluate origin of cSHMT, Tbx3 and utrophinin plasma, we immunoblotted aliquots of cell extracts, plasma andmixture of cell extracts with plasma (ratio 1:3, cell extract to plasma,respectively; 1 h incubation) with respective specific antibodies, asindicated in FIGS. 2, 3 and 4.

Correlation analysis of cSHMT expression and survival of patients withovarian cancer was performed by univariate Kaplan-Meier survivalanalysis¹² using the Statistica software (v.6.0, StatSoft, Inc., Tulsa,USA). We used this method for evaluation of cumulative proportionaloverall survival of ovarian cancer patients, who were divided into twogroups according to the level of expression of cSHMT in plasma.

Results Preparation of Samples and Two-Dimensional Gel Electrophoresisof Plasma

To ensure reliability in sample preparation, we established a procedurefor collection of human plasma as described previously herein. Thisprocedure is based on ethanol precipitation of aliquots of freshlycollected sodium citrated plasma. Dried protein precipitates can bestored for further analysis (FIG. 1A). This procedure is also compatiblewith 2D-GE. A procedure similar to the one developed by us was approvedby the Human Plasma Initiative group of the Human Proteome Organization(www.hupo.org).

Collected plasma samples were subjected to 2D-GE (FIG. 1B). Theinterference with protein migration due to serum albumin andimmunoglobulins was observed only upon loading of gels with more than150 μg of proteins. Upon loading of 100 μg of proteins or less,perturbation of the protein migration was limited. Accordingly, plasmawas not depleted from the albumin and immunoglobulins. Elimination ofthe depletion procedure increased reproducibility of sample preparation,as additional steps in plasma manipulation were excluded. The simplicityof the developed sample preparation procedure also facilitates its useat the clinic.

Twenty-one samples from breast cancer patients, 14 samples from ovariancancer patients, and 10 samples from individuals without cancer weresubjected to 2D-GE. Images of silver-stained gels were analyzed, and onaverage 700 protein spots were observed in each gel. The variationbetween the numbers of protein spots in gels of the same sample wasbelow 10% of the total number of spots. If protein spots appeared in 2Dgels of one or only a few samples, it was considered as individualvariation, and spots were not considered for analysis. Only proteinspots which were present and/or changed their expression in more than80% of samples representing a single clinical group of patients wereconsidered for further analysis. In total, 45 2D gels were generated,with repeating of 2D-GE of the same samples. These repeats wereperformed to exclude experimental variations and to select for furtheranalysis only reproducibly detected protein spots.

Three proteins with different expression levels in plasma of cancer andnoncancer individuals were identified as cSHMT, Tbx3 and utrophin (FIG.1C; Table 2). It should be noted that migration positions in 2D gels ofidentified proteins indicated lower molecular masses than would beexpected for the full-length proteins (Table 2). By peptide massfingerprinting, a 52 kDa isoform of Tbx3 was identified, but in 2D gelsit was observed as a 17 kDa protein. We observed, however, that thepeptides which were used for identification originated from the centralpart of the protein. This indicated that we had observed a truncatedform of Tbx3 and that for a validation study, an antibody to theinternal region of Tbx3 had to be used. The full-length utrophin wasdescribed as a large 395 kDa protein, but a number of splice forms oflow molecular mass have been identified.¹³ For a validation study, anantibody which recognizes an epitope in the N-terminal part of utrophinwas used. For cSHMT, lack of the N-terminal peptides also indicatedtruncation which resulted in a protein of lower molecular mass, andwhich corresponded to the protein spot observed in 2D gels (FIG. 1).This prompted us to use an antibody to an epitope in the C-terminal partof cSHMT. cSHMT, Tbx3 and utrophin have been described as intracellularproteins,¹³⁻¹⁵ and their detection in plasma indicates their releasefrom cells and limited proteolysis.

Thus, we developed a protocol for collection of plasma, performed 2D-GEand image analysis of samples from cancer and noncancer individuals, anddiscovered aberrant expression of truncated cSHMT, Tbx3 and utrophin inplasma from breast and ovarian cancer patients.

Aberrant Expression of cSHMT, Tbx3 and Utrophin in Plasma

To confirm and further explore aberrant expression of cSHMT, Tbx3 andutrophin, we performed immunoblotting analysis of plasma samples from alarge cohort of patients (Table 1). The validation study was ofimportance, as not all tryptic peptides from the selected protein spotswere used for identification of cSHMT, Tbx3 and utrophin by peptide massfingerprinting. It is thus possible that other proteins could comigratein the same spot. The validation by an alternative technique wasessential also because peptide mass fingerprinting provided anindication, but did not give 100% confidence of identification. For thevalidation study, plasma samples were prepared according to the protocolthat was used for 2D-GE. However, we used 1D-GE, which allowedcomparison of many samples in one gel (FIGS. 2, 3, and 4).

Specific antibodies to cSHMT recognized a protein band migrating inplasma samples at the position of 16.5 kDa (FIG. 2A). To controlspecificity, we found that preincubation of antibodies with specificblocking peptide blocked recognition of this band in a whole-cellextract of MCF7 human breast cancer cells (FIG. 2B). We explored whetherincubation of cellular cSHMT with plasma would result in generation oflow-molecular-mass product. cSHMT was first detected in COS7 cellextract, and this extract was incubated with fresh plasma (FIG. 2C). Weused COS7 cell extract and fresh plasma from a healthy donor, asethanol-precipitated plasma from patients might have impairedproteolytic activity. Incubation of COS7 cell extracts with fresh plasmaled to generation of a cSHMT-specific band of low molecular mass (FIG.2C). This suggested that the low-molecular-mass cSHMT-specific band isthe result of limited proteolysis.

cSHMT-specific protein expression in plasma from the group of healthyand noncancer patients was observed with weak intensity in 3 cases outof 31 (FIGS. 2 and 5). Expression of cSHMT was observed in the samplesof patients with stage I of ovarian cancer. At stages II, III and IV ofthe disease, the frequency of cSHMT detection was similar to that ofstage I, or higher (for stage IV). Expression of cSHMT was alsostronger, as compared to samples from the control group (FIG. 5A). Morethan 5-fold more frequent and stronger expression of cSHMT was observedfor the breast cancer patients with stage I of the disease, as comparedto the control samples (FIG. 5B). The frequency of cSHMT detection ingroup of patients with stage II and stages III-IV of breast cancer wassimilar to that observed for patients with stage I (FIG. 5B).Specificity of cSHMT expression was found to be 90%, and sensitivitieswere 81% for breast cancer and 74% for ovarian cancer.

Antibodies specific to Tbx3 detected proteins of molecular masses of 80kDa, 62 kDa and approximately 20 kDa, upon expression of Tbx3 in 293Tcells (FIG. 3A). As the Tbx3 construct contained an HA-tag, we couldconfirm the specificity of the anti-Tbx3 antibodies by reprobing thesame membrane with anti-HA antibodies. Detection of Tbx3 was abrogatedby the specific blocking peptide (FIG. 3A). We explored whetherincubation of a cellular extract with plasma would result in generationof a low-molecular-mass product. Tbx3 was first detected in MCF7 cellextract (FIG. 3B), and this extract was incubated with fresh plasma, asdescribed for FIG. 2C. We observed generation of a Tbx3-specific 62 kDaband (FIG. 3B), suggesting that it is the product of a limitedproteolysis.

In plasma samples, we observed a strong Tbx3-specific signal from aprotein migrating at 62 kDa (FIG. 3C), and a much weaker signal from aprotein migrating at approximately 20 kDa. As both 62 kDa and 20 kDaproteins may be products of limited proteolysis of the full-length Tbx3(FIGS. 3A and B), we studied the expression of the 62 kDa form of Tbx3.It is possible that the 62 kDa form of Tbx3 comigrated with serumalbumin, and therefore was not detected in 2D-GE, while in theimmunoblotting experiment the 62 kDa form was the most prominent. Weobserved weak expression of Tbx3 in 30% of samples from noncancerouspatients (FIG. 3C). In plasma samples from cancer patients, Tbx3 wasdetected in all cases of stage I of ovarian cancer, and in 80% ofpatients with breast cancer (FIGS. 5C and D). Tbx3 was also detected inall samples from patients with advanced breast cancers (FIG. 5D). In allcancer cases, the levels of Tbx3 expression were elevated (FIGS. 5C andD). Specificity of Tbx3 expression was 68%, and sensitivities were 98%for breast cancer and 90% for ovarian cancer cases.

Immunoblotting with antibodies specific to utrophin identified a strongprotein band of molecular mass approximately 30 kDa in the plasmasamples of cancer patients (FIG. 4A). This migration positioncorresponds to the migration position of the protein spot in 2D gels, inwhich utrophin was identified. As we explored for cSHMT and Tbx3 (FIGS.2C and 3B), we also studied generation of a 33 kDa utrophin-specificband (FIG. 4B). We observed a strong generation of 33 kDa bandrecognized by antiutrophin antibodies upon incubation of cell extractwith plasma (FIG. 4B).

We observed weak expression of utrophin in a number of plasma samplesfrom noncancer patients (FIG. 4C). However, in samples from cancerpatients expression of utrophin was significantly stronger and morefrequent (FIGS. 5E and F). In the breast cancer samples, utrophin wasdetected in all samples of stage I, with increased level of expressionin more than 75% of the cases (FIGS. 5E and F). In the ovarian cancersamples, frequency of utrophin expression increased to more than 80% ofthe cases, with an increased level of protein expression (FIG. 5E).Specificity of utrophin expression was 83%, and sensitivities were 72%for breast cancer and 100% for ovarian cancer cases.

cSHMT, Tbx3 and utrophin have been described as intracellularproteins.¹³⁻¹⁵ Their detection in plasma, as truncated proteins,suggests that they were released from cells. The molecular mechanisms oftumorigenesis may vary between tumor cells of the same type of cancer.This explains why expression patterns of a single separately takenprotein (FIGS. 2, 3 and 4) did not correlate to 100% with a type ofcancer, or a stage of disease. Thus, combined patterns of expression ofcSHMT, Tbx3 and utrophin in plasma may be included in multifactorialprediction of the early stages of cancer.

Twenty-four-month overall survival of ovarian cancer patients was higherin the group with negative and low expression of cSHMT in plasma (4patients died out of 12 followed), as compared to the patients withmoderate and strong expression of cSHMT (5 patients died out of 7followed; FIG. 6). During the follow-up period, 4 breast cancer patientswith stage III died, while the other 75 patients were alive by Novemberof 2004, i.e. more than 24 months after the initial diagnosis.

In a follow-up study, the overall survival of 39 ovarian cancer patientsover a 48-month period was correlated with cSHMT expression in plasma.As can be seen in FIG. 7, higher levels of cSHMT expression correspondedto much lower rates of survival. After 48 months, the survival rate ofpatients with low levels of expression was 80%, whereas after only 36months, the survival rate of patients with moderate expression was onlyabout 15%. In addition, the prognostic factor for cSHMT expression andsurvival of ovarian cancer patients was determined by multivariateanalysis (Cox regression model). By this analysis also (see Table 3), astrong correlation was demonstrated between level of cSHMT expression inplasma and overall survival of ovarian cancer patients.

Strong correlations between Tbx3 and utrophin expression in plasma andoverall survival of breast cancer patients were also observed.

The overall survival of 50 breast cancer patients over a 48-month periodwas correlated with Tbx3 expression in plasma. As can be seen in FIG. 8,the survival rate of patients with moderate levels of expression wasdramatically lower than the rate for patients with low levels ofexpression.

Similarly, the overall survival of 55 breast cancer patients over a48-month period was correlated with utrophin expression in plasma.Again, as can be seen in FIG. 9, the survival rate of patients withmoderate levels of expression was dramatically lower than the rate forpatients with low levels of expression.

In addition, the prognostic factors for Tbx3 and utrophin expression inplasma and survival of breast cancer patients were determined bymultivariate analysis (Cox regression model). By this analysis also (seeTable 4), strong correlations were demonstrated both 1) between levelsof Tbx3 expression and breast cancer survival and 2) between levels ofutrophin expression and breast cancer survival.

The data demonstrate a correlation between expression in plasma of eachof the three markers and truncated forms thereof and the stage ofcancer, as well as a correlation between expression of the markers andaggressiveness of the disease.

TABLE 1 Expression (%) of cSHMT, Tbx3, utrophin in plasma of control(A), and ovarian and breast cancer patients (B) A) cSHMT Tbx3 utrophinPatient Diagnosis expression, % expression, % expression, % A Healthy 0— — B Healthy 0 — — C cervical epithelium dysplasia 0 — — D cervicalepithelium dysplasia 0 — — E cervical epithelium dysplasia 0 — — Fcervical epithelium dysplasia 0 — — G serous cyst of left ovary 0 — — Hserous cyst of left ovary 0 — — I fibromyoma of uterus 0 — — J serouscystadenoma of ovary 0 — — K fibroadenoma of right breast 0 — — Lfibroadenoma of left breast 0 — —  2 serous cyst of ovary 0 0 0  4dermoid cyst, adenofibroma of ovary 0 0 0  5 adenocystous polyp ofendometrium 0 9 0  10 fibromyoma of uterus 0 10 0  13 fibrous mastopathyof breast 0 7 18  17 folioid fibromyoma of breast 0 10 0  18proliferative mastopathy with sclerotic adenosis 0 13 0  21 endometrioidcyst of ovary 0 12 0  23 fibrous mastopathy, chronichal mastitis 0 10 8 29 Healthy 0 9 18  33 fibroadenomatosis with focal sclerotic adenosis 08 26 of breast  52 mucinous cystadenoma of ovary 0 10 36  25 serouscystadenoma of ovary 0 10 21  47 fibroadenoma of breast 0 15 0  79follicular cyst of ovary, chronichal salpingitis 5 13 —  82cylioepithelial cyst of ovary 7 14 0 104 serous cyst of ovary 4 11 0 108interrupted tubal pregnancy 10 8 0 117 lipoma of breast 8 9 0 B) cSHMTTbx3 utrophin Patient Diagnosis pT pN pM pG stage expression, %expression, % expression, % a ovarian cancer 3C 0 0 III 26 — — b ovariancancer 2 0 1 IV 25 — — c ovarian cancer 2 0 0 II 14 — — d ovarian cancer1C 0 0 2 IC 23 — — e ovarian cancer 1C 0 0 2 IC 10 — — f ovarian cancer3C 0 0 IIIC 32 — — g ovarian cancer 3C 0 0 IIIC 17 — —  1 ovarian cancer3 0 0 III 31 11 0  3 ovarian cancer 3 0 0 III 22 19 0  20 ovarian cancer2 0 0 II 35 23 0  27 ovarian cancer 2 0 1 IV 17 15 0  39 ovarian cancer2 0 0 II 12 13 41  40 ovarian cancer 3A 0 0 IIIA 93 12 24  41 ovariancancer 2 0 0 II 66 12 27  48 ovarian cancer 3 0 0 III 29 15 —  50ovarian cancer 3A ∘ 0 1 III 18 16 —  51 ovarian cancer x x 1 IV 63 18 — 53 ovarian cancer 2 0 0 II 29 18 67  55 ovarian cancer x x 1 IV 6 15 51 57 ovarian cancer x x 1 IV 33 57 66  78 ovarian cancer x x x x 12 0 57 80 ovarian cancer 1A 0 0 1 IA 21 25 61  83 ovarian cancer 3 0 0 III 0 758  84 ovarian cancer 3C 0 0 III 0 10 57  86 ovarian cancer 2B 0 0 2 IIB7 28 49  88 ovarian cancer 2B 0 0 II 11 — 32  89 ovarian cancer 2B 0 0II 17 57 36  90 ovarian cancer 3B 0 0 1 III — — 32  93 ovarian cancer 30 1 IV 55 71 54 107 ovarian cancer 3 0 0 III 48 79 — 116 ovarian cancer3C 0 0 IIIC 23 28 — 118 ovarian cancer 1A 0 0 1 IA 0 41 — 120 ovariancancer 2B 0 0 II 0 45 40 123 ovarian cancer 3C 0 0 III 25 60 43 134ovarian cancer 3 0 0 III 14 33 30 135 ovarian cancer 2A 0 0 II 0 36 23141 ovarian cancer 1C 0 0 IC 0 33 — 142 ovarian cancer 1C 0 0 IC 54 54 —143 ovarian cancer 3C 0 0 III 0 — 44 g1 breast cancer 4 2 0 III 43 — —g2 breast cancer 1 0 0 I 62 — — g3 breast cancer 4 2 0 IV 18 — — g4breast cancer 2 0 0 II 22 — — g5 breast cancer 2 0 0 II 34 — — g6 breastcancer 2 0 0 II 21 — — g7 breast cancer 1 0 0 II 24 — — g8 breast cancer2 1 0 II 35 — — g9 breast cancer 1 0 0 I 67 — — g10 breast cancer 2 0 03 II 43 — — g11 breast cancer 2 0 0 3 IIA 15 — — g12 breast cancer x x 1IV 13 — — g14 breast cancer 1 0 0 I 21 — — g15 breast cancer 1 2 0 3 III16 — — g16 breast cancer 2 0 0 II 6 — — g17 breast cancer 2 0 0 2 II 7 —— g19 breast cancer 2 0 1 IV 0 — — g20 breast cancer 1 0 0 1 I 0 — — g21breast cancer is 0 0 0(Cr is) 6 — — g22 breast cancer 2 0 0 3 II 4 — — 6 breast cancer 2 1 0 IIA — — 33  7 breast cancer 2 1 0 IIA — — 46  8breast cancer 2 1 0 IIA 6 52 —  11 breast cancer 1 0 0 I 14 — —  12breast cancer 2 1 0 IIB 17 — 75  14 breast cancer 2 1 0 III 17 20 85  15breast cancer 1 1A 0 2 II 27 18 52  16 breast cancer 2 0 0 IIA 38 20 20 22 breast cancer 2 1 0 2 IIB 24 20 18  30 breast cancer 2 2 0 3 IIIA 1930 21  31 breast cancer 1 0 0 1 I 51 9 18  32 breast cancer 2 1 0 IIB 2015 15  34 breast cancer x x x x 52 40 17  35 breast cancer x x x 2 x 2419 20  36 breast cancer 4 0 0 3 IV 22 42 14  42 breast cancer x x x x 28116 15  44 breast cancer 2 1 0 2 II 13 150 18  45 breast cancer 2 1 0IIIA 0 92 21  46 breast cancer x x x 2 x 23 136 21  58 breast cancer 2 10 2 III — 57 19  59 breast cancer x x x x — 51 —  61 breast cancer 2 0 02 II 0 57 30  62 breast cancer 2 0 0 2 II — 31 32  64 breast cancer 2 00 II — 36 46  65 breast cancer 2 0 0 2 II — 19 52  66 breast cancer 2 10 2 III — 27 44  69 breast cancer is 0 0 0(Cr is) 12 24 53  70 breastcancer 2 0 0 2 II 17 25 36  71 breast cancer 2 0 0 2 II 9 — 17  72breast cancer 2 0 0 2 II 14 — 58  73 breast cancer 2 0 0 2 II 35 62 28 96 breast cancer 3 0 0 3 III — 61 17  97 breast cancer 2 0 0 IIA — 51 5 98 breast cancer 2 0 0 IIA — 83 6  99 breast cancer 2 0 0 IIA — 23 6101 breast cancer 2 2 0 IIIA — 22 — 102 breast cancer 3 1 0 2 IIIA 26 2563 105 breast cancer 3 0 0 2 IIB 9 12 55 106 breast cancer 2 1 0 2 IIB —42 66 109 breast cancer 3 3 0 2 IIIB — 36 64 110 breast cancer 2 1 0 IIB21 18 60 111 breast cancer 2 0 0 IIA 18 32 71 113 breast cancer 2 1 0IIB 30 37 71 114 breast cancer 4 2 0 3 IIIB 53 106 104 115 breast cancerx x x 2 X 39 61 106 119 breast cancer 2 0 0 2 IIA — 52 71 121 breastcancer 3 1 0 2 IIIB — 58 66 122 breast cancer 1 1 0 2 III 76 — 79 124breast cancer 2 1 0 III 51 — 93 125 breast cancer 2 1 0 2 III 32 — 73126 breast cancer 2 1 0 2 III 18 91 80 127 breast cancer 4 0 0 2 IIIB 085 48 128 breast cancer 1 0 0 I 27 45 47 129 breast cancer 2 1 0 2 III31 32 37 130 breast cancer 2 1 0 III 42 39 42 132 breast cancer 2 0 0IIA 45 30 43 133 breast cancer 1 0 0 I 52 41 52 138 breast cancer 2 1 02 III 79 89 60 139 breast cancer 2 1 0 2 III 63 60 76 pT = tumor size pN= lymph node metastasis pM = metastasis pG = tumor grade

TABLE 2 Identification of cSHMT, Tbx3 and utrophin in protein spots.Theoretical Experimental Sequence NCBI value c) value c) Spot Z coverageaccession Mr Mr No. a) Protein b) Probability b) value b) (%) b) numberb) pI (kDa) pI (kDa) s502 Cytosolic serine 9.9e−0.01 0.72 12 Y14487 6.938.7 6.0 18.0 hydroxymethyl- transferase s515 Tbx3 9.3e−0.01 0.58 12AF002228 8.4 52.1 5.3 17.0 s385 Utrophin 1.0e+000 1.23 13 X69086 5.968.8 5.5 30.0 a) Spot numbers are as annotated by the image analysis. b)Protein names, probability, Z, sequence coverage and NCBI accessionnumbers are as obtained in a search of NCBI database with ProFoundsearch engine. c) Theoretical and experimental values of pI andmolecular mass are indicated. Experimental values were calculated frommigration positions of proteins in 2D gels.

TABLE 3 Independent prognostic factors of the survival of ovarian cancerpatients (n = 39), multivariate analysis (Cox regression model).Prognostic factor P cSHMT expression in plasma 0.013 Tbx3 expression inplasma 0.560 Utrophin expression in plasma 0.334 χ² = 26.79, p = 0.0015.(See also annotations to Table 4)

TABLE 4 Independent prognostic factors* of the survival of breast cancerpatients (n = 76), multivariate analysis (Cox regression model).Prognostic factor P** cSHMT expression in plasma 0.159 Tbx3 expressionin plasma 0.019 Utrophin expression in plasma 0.025 χ² = 20.72, p =0.0232 *Independent prognostic value for overall survival of breastcancer patients was calculated for cSHMT, Tbx3 and utrophin expressionin blood plasma and the following parameters: pT 1-4 (tumor size), pN0-2 (lymph node metastasis), M 0-1 (metastasis), pG 1-3 (tumor grade),stage (I-IV), tumor histology type (lobular and ductal), age (below andover 40). **P, prognostic factor significance; in bold—significantvalues.

As shown by the disclosure herein, it has been discovered that increasedexpression in plasma of cSHMT, Tbx3, utrophin and truncated formsthereof, either alone or in combinations, has a strong correlation withincreased incidence of ovarian and breast cancer, as well as decreasedtime and chance of survival for cancer patients.

The present application describes not only the discovery of thesecorrelations but also the discovery of a number of truncated forms ofcSHMT, Tbx3 and utrophin. The present invention is directed not only tothe specific truncated forms mentioned herein but to all truncated formsof these markers that may be produced in conjunction with the onset ofovarian and/or breast cancer. These truncated forms are readilyvisualized, identified and quantitated by the techniques describedherein. Furthermore, the actual sequences of the truncated forms mayreadily be derived by one of skill in the art by employment of standardtechniques of peptide and amino acid analysis (see, for example,Proteins and Proteomics, Richard J. Simpson, Cold Spring HarborLaboratory Press (2003) and Modern Protein Chemistry: Practical Aspects,G. C. Howard and W. E. Brown, Eds., CRC Press, Boca Raton, Fla. (2002))and the prior knowledge of the primary structures of the full-lengthforms of the proteins.

The present invention further encompasses antibodies raised against thefull-length and truncated forms of the protein markers, which antibodiesare necessary for the practice of the diagnostic and prognostic methodsdescribed herein. Such antibodies may be produced by any of thetechniques well known to one of skill in the art. (See, for example,Monoclonal Antibodies: Methods and Protocols, R. Rose and M. Albitar,Eds., Humana Press, 1st Edition (2007) and Antibodies: A LaboratoryManual, Harlow and Lane, Cold Spring Harbor Laboratory Press (2003).)Although monoclonal antibodies are preferred for the practice of theinvention, the invention also encompasses polyclonal antibodies ofsuitable specificity.

The discovery of the correlations set forth herein and the discovery ofthe truncated forms of the protein markers also provide the means fordiagnosing ovarian and/or breast cancer and for predicting longevity andchance of survival of patients with these cancers. Accordingly, anotheraspect of the present invention are the diagnostic and prognosticmethods disclosed herein in connection with ovarian and/or breastcancer. These methods comprise the steps of a) obtaining a plasma samplefrom a patient; b) testing among the proteins in the sample for thepresence and amount of one or more of cSHMT, Tbx3, utrophin andtruncated forms thereof; and either, for diagnostic purposes, c) usingthe level(s) of the marker(s) detected in a patient of unknown status todetermine the likelihood or not of the patient having ovarian and/orbreast cancer or, for prognostic purposes, d) using the level(s) of themarker(s) detected in a patient already known to have ovarian and/orbreast cancer to determine the optimal regimen of treatment, predict thepatient's response to the treatment and to predict the likelihood orduration of survival.

Still further, the invention encompasses kits comprising one or moreantibodies generated against cSHMT, Tbx3 and utrophin, and againsttruncated forms thereof associated with the onset of ovarian and/orbreast cancer, which kits are useful in the diagnosis and prognosis ofovarian and/or breast cancer.

The present invention has added three proteins, and truncated formsthereof, to the list of markers which can be used for creation of adiagnostic and prognostic microarray in connection with ovarian and/orbreast cancer. As borne out by the showing herein, the novel markers ofthe present invention may be used either singularly or, in amultiparameter diagnostic or prognostic approach, in variouscombinations. It is further expected that one or more of the inventivemarkers can be used in combination with other, previously known markersin a multiparameter diagnostic or prognostic approach. The additionalmarkers may be proteinaceous or not and may have their origins in eitherplasma or tissue. Nonlimiting examples of such markers are CA125,CA15-3, CEA, RS/DJ1, apolipoprotein A1, transthyretin, inter-α trypsininhibitor heavy chain H4, haptoglobin-1 and kallikrein; lysophosphatidicacid and DNA^(3,20) and estrogen receptors such as ErbB2/neu andKi-67.²¹

The invention is thus also directed to microarrays of proteins and othermarkers for use in the diagnosis and/or prognosis of ovarian and/orbreast cancer. These microarrays comprise one or more markers selectedfrom cSHMT, Tbx3, utrophin and truncated forms thereof in combinationwith one more previously known markers as exemplified above.

REFERENCES

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Legends for FIGS. 1-6

FIG. 1. Detection of cSHMT, Tbx3 and utrophin in two-dimensional gels ofplasma samples. (A) The scheme of the study is presented. Plasma wasprepared from collected blood, and proteins were precipitated withethanol. 100 μg of plasma proteins were separated by 2D-GE.Differentially expressed proteins were identified by MALDI TOF MS.Samples from a large cohort of patients were tested by immunoblottingwith antibodies specific to selected proteins. (B) An image of a 2D gelis shown to illustrate quality of protein resolution. The gel representsa control sample from a noncancer patient. The pH gradient is indicatedon the top of the gel, and migration positions of molecular mass markersare indicated on the side of the gel. Areas of migration of cSHMT, Tbx3and utrophin are indicated by squares. (C) Areas of migration of cSHMT(upper panels), Tbx3 (middle panels) and utrophin (lower panels) in 2Dgels of control (left row), ovarian cancer (middle row) and breastcancer (right row) samples are shown. Protein spots containing cSHMT,Tbx3 and utrophin are indicated by circles. Arrowheads indicatemigration positions of reference protein spots.

FIG. 2. Detection of cSHMT in plasma samples by immunoblotting. (A)Samples were prepared as for 2D-GE, and were subjected to 1D SDS-PAGE,transferred onto membrane and immunoblotted with anti-cSHMT antibodies,as described earlier herein. Migration positions of a protein withmolecular mass which corresponds to the cSHMT identified in 2D gels areindicated by arrows in upper panels. IgG-specific protein bands,detected in all samples after reprobing the same membranes withantihuman IgG are indicated by arrows in lower panels. (B) Specificblocking peptide abrogated recognition of a protein migrating at theposition of 16.5 kDa. The whole-cell extract from MCF7 cells wassubjected to SDS-PAGE and immunoblotting with antibodies to cSHMT whichwere preincubated or not with blocking peptide, as indicated. Migrationpositions of a protein with molecular mass which corresponds to thecSHMT identified in 2D gels is indicated by an arrow. (C) Alow-molecular-mass cSHMT-specific band was detected after incubation ofCOS7 cell extract with fresh plasma from a healthy individual(COS7+plasma). Aliquots of cell extract (COS7), plasma only (plasma),and mixture of cell extract with plasma in the ratio 1:3, respectively(1:3; 1:3 precipitate) were immunoblotted with anti-cSHMT antibodies.The “1:3 precipitate” fraction shows the mixture of cell extract andplasma prepared according to the plasma preparation protocol describedearlier herein. The arrow shows a cSHMT-specific protein band whichincreased after incubation of the cell extract with plasma. Migrationpositions of molecular mass markers are indicated on the side of imagesAnnotations of samples in panel (A) are as in Table 1. Representativeexperiments with randomly selected samples out of 4 (A) and 3 (B, C)performed are shown.

FIG. 3. Detection of Tbx3 in plasma samples by immunoblotting. (A)Specific blocking peptide abrogated recognition by antibodies specificto Tbx3 of prominent proteins with molecular masses of 80 kDa and 62kDa, and of a less strongly expressed protein of 20 kDa. Arrows indicatethe proteins. (B) A 62 kDa Tbx3-specific band was detected afterincubation of MCF7 cell extract with fresh plasma from a healthyindividual (MCF7+plasma). Aliquots of cell extract (MCF7), plasma only(plasma), and mixture of cell extract with plasma in the ratio 1:3,respectively (1:3; 1:3 precipitate) were immunoblotted with anti-Tbx3antibodies. The “1:3 precipitate” fraction shows the mixture of cellextract and plasma prepared according to the protocol described earlierherein. The arrow shows a Tbx3-specific protein band which increasedafter incubation of the cell extract with plasma. Migration positions ofmolecular mass markers are indicated on the side of images. (C) Plasmasamples were prepared as for 2D-GE, and were subjected to 1D SDS-PAGE,transferred onto membrane and immunoblotted with anti-Tbx3 antibodies,as described earlier herein. Migration positions of a protein withmolecular mass which corresponds to the truncated Tbx3 are indicated byarrows in upper panels. IgG-specific protein bands, detected in allsamples after reprobing the same membranes with antihuman IgG, areindicated by arrows in lower panels. Annotations of samples are as inTable 1. Representative experiments with randomly selected samples outof 4 (C) and 2 (A, B) performed are shown.

FIG. 4. Detection of utrophin in plasma samples by immunoblotting. (A)Expressions of utrophin truncated isoform in plasma samples fromcontrol, ovarian and breast cancer groups are shown. The arrow indicatesmigration position of the utrophin isoform. The arrowhead indicatesmigration position of a nonspecific protein. Migration positions ofmolecular mass markers are indicated on the side of images. (B) A 33 kDautrophin-specific band was detected after incubation of 293T cellextract with fresh plasma from a healthy individual (293T+plasma).Samples were prepared as in FIG. 2C. Notably, aliquots of cell extract(293T), plasma only (plasma), and mixture of cell extract with plasma inthe ratio 1:3, respectively (1:3; 1:3 precipitate) were immunoblottedwith anti-utrophin antibodies. The “1:3 precipitate” fraction shows themixture of cell extract and plasma prepared according to the protocoldescribed earlier herein. The arrow shows a utrophin-specific proteinband which increased after incubation of the cell extract with plasma.(C) Plasma samples were prepared as for 2D-GE, and were subjected to 1DSDS-PAGE, transferred onto membrane and immunoblotted with antiutrophinantibodies, as described earlier herein. Migration positions of aprotein with molecular mass which corresponds to the utrophin truncatedisoform are indicated by arrows in upper panels. IgG-specific proteinbands, detected in all samples after reprobing the same membranes withantihuman IgG are indicated by arrows in lower panels. Annotations ofsamples are as in Table 1. Representative experiments with randomlyselected samples out of 4 (C), 3 (A) and 2 (B) performed are shown.

FIG. 5. Expression of cSHMT, Tbx3 and utrophin in samples of patientswith various stages of cancer. Expression of cSHMT, Tbx3 and utrophinwas normalized to intensities of non-specific bands which were presentin all samples, and was calculated as a relative intensity as describedearlier herein. Distributions of relative intensities of proteinexpressions for various stages of patients with ovarian (A, C, E) andbreast (B, D, F) cancers are indicated. Expressions of cSHMT (A, B),Tbx3 (C, D) and utrophin (E, F) are shown. For cSHMT, a relativeintensity of the specific signal to the IgG band of less than 25% wasevaluated as weak (light grey bar), between 26% and 50% as medium(medium grey bar), and higher than 50% as high (dark grey bar). ForTbx3, a relative intensity of up to 40% was evaluated as weak, between41% and 60% as medium, and higher than 60% as high. For utrophin, arelative intensity of up to 40% was evaluated as weak, between 41% and70% as medium, and higher than 70% as high. Stages of cancers areindicated in panels. Cutpoints are defined as provided earlier herein.

FIG. 6. Expression of cSHMT correlated with shorter survival of ovariancancer patients. Comparison of the cumulative overall survival within a24-month follow-up period of patients of groups with low (<25%; 12patients, 4 died) and moderate/high (>25%; 7 patients, 5 died)expression of cSHMT in plasma was performed by univariate Kaplan-Meieranalysis¹² (p<0.01).

1. (canceled)
 2. A protein marker selected from the group consisting ofcSHMT, Tbx3, utrophin and a truncated form of any one of these proteins.3. An antibody raised against, a protein selected from the groupconsisting of cSHMT, Tbx3, utrophin and a truncated form of any one ofthese proteins.
 4. A kit for the diagnosis of ovarian and/or breastcancer comprising one or more antibodies raised against protein markersselected from cSHMT, Tbx3, utrophin and truncated forms of any of thesemarkers.
 5. A kit for the prognosis of ovarian and/or breast cancercomprising one or more antibodies raised against protein markersselected from cSHMT, Tbx3, utrophin and truncated forms of any of thesemarkers. 6-8. (canceled)
 9. A microarray of proteins and other markersfor use in the diagnosis and/or prognosis of ovarian and/or breastcancer which comprises one or more markers selected from cSHMT, Thx3,utrophin and truncated forms thereof in combination with one or morepreviously known markers.
 10. The microarray according to claim 9wherein the one or more previously known markers are selected from thegroup consisting of CA125, CA15-3, CEA, RS/DJ1, apolipoprotein A1,transthyretin, inter-α trypsin inhibitor heavy chain H4, haptoglobin-1,kallikrein, lysophosphatidic acid, DNA, ErbB2/neu and Ki-67.
 11. The kitaccording to claim 4, wherein the kit is used to test a plasma sampleobtained from a patient for the presence and amount of one or more ofthe markers and wherein 1) observation of aberrant levels of one or moreof the markers identifies the patient as likely having ovarian and/orbreast cancer or 2) observation of normal or no expression of themarkers identifies the patient as free of those cancers.
 12. The kitaccording to claim 5, wherein the kit is used to test a plasma sampleobtained from a patient for the presence and amount of one or more ofthe markers and wherein the amounts of aberrant levels of any or all ofthe markers are used for determining the optimal treatment regimen forthe patient, for predicting the patient's response to the treatment andfor predicting the likelihood of duration of survival of the patient.